The enzyme linked immunosorbent spot (ELISPOT) assay has become widely utilized as a tool to study cellular and humoral immune responses in vitro. ELISPOT has been used to determine the frequency of specific CD4+ and CD8+ T cell responses to self, tumor, viral, bacterial and other antigens. Secretion of a range of cytokines (IFN-γ, TGF-β, TNF-α, IL-4, IL-5, IL-6, IL-10, IL-12), β-chemokines (MIP-1α, MIP-1β, RANTES) and cytotoxins (granzymes) may be measured. The sensitivity of the ELISPOT assay for detecting CD8+ T cell responses has been estimated to be at least 1 log10 higher than traditional limiting dilution analysis or bulk 51Cr release assay.
Use of autologous dendritic cells as antigen-presenting cells may augment the sensitivity of ELISPOT. However, this method requires large amounts of fresh peripheral blood and in vitro maturation of monocytes over a period of 5 to 7 days. Thus, although the sensitivity and technical ease of the ELISPOT assay make it a useful alternative to traditional analytical methods, there remains a need for increased sensitivity in detection of low frequency antigen-specific T cell responses. The present invention addresses this need.
Literature
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